Structural und functional characterization of Rubisco assembly chaperones

In the present study, the structure and mechanism of two assembly chaperones of Rubisco, Raf1 and RbcX, were investigated. The role of Raf1 in Rubisco assembly was elucidated by analyzing cyanobacterial and plant Raf1 with a vast array of biochemical and biophysical techniques. Raf1 is a dimeric protein. The subunits have a two-domain structure. The crystal structures of two separate domains of Arabidopsis thaliana (At) Raf1 were solved at resolutions of 1.95 Å and 2.6–2.8 Å, respectively. The oligomeric state of Raf1 proteins was investigated by size exclusion chromatography connected to multi angle light scattering (SEC-MALS) and native mass spectrometry (MS). Both cyanobacterial and plant Raf1 are dimeric with an N-terminal domain that is connected via a flexible linker to the C-terminal dimerization domain. Both Raf1 poteins were able to promote assembly of cyanobacterial Rubisco in an in vitro reconstitution system. The homologous cyanobacterial system resulted in very high yields of active Rubisco (>90%), showing the great efficiency of Raf1 mediated Rubisco assembly. Two distinct oligomeric complex assemblies in the assembly reaction could be identified via native PAGE immunoblot analyses as well as SEC-MALS and native MS. Furthermore, a structure-guided mutational analysis of Raf1 conserved residues in both domains was performed and residues crucial for Raf1 function were identified. A new model of Raf1 mediated Rubisco-assembly could be proposed by analyzing the Raf1-Rubisco oligomeric complex with negative stain electron microscopy. The final model was validated by determining Raf1-Rubisco interaction sites using chemical crosslinking in combination with mass spectrometry. Taken together, Raf1 acts downstream of chaperonin-assisted Rubisco large subunit (RbcL) folding by stabilizing RbcL antiparallel dimers for assembly into RbcL8 complexes with four Raf1 dimers bound. Raf1 displacement by Rubisco small subunit (RbcS) results in holoenzyme formation. In the second part of this thesis, the role of eukaryotic RbcX proteins in Rubisco assembly was investigated. Eukaryots have two distinct homologs of RbcX, RbcX-I and RbcX-II. Both, plant and algal RbcX proteins were found to promote cyanobacterial Rubisco assembly in an in vitro reconstitution system. Mutation of a conserved residue important for Rubisco assembly in cyanobacterial RbcX also abolished assembly by eukaryotic RbcX, underlining functional similarities among RbcX proteins from different species. The crystal structure of Chlamydomonas reinhardtii (Cr) RbcX was solved at a resolution of 2.0 Å. RbcX forms an arc-shaped dimer with a central hydrophobic cleft for binding the C-terminal sequence of RbcL. Structural analysis of a fusion protein of CrRbcX and the C-terminal peptide of RbcL suggests that the peptide binding mode of CrRbcX may differ from that of cyanobacterial RbcX. RbcX homologs appear to have adapted to their cognate Rubisco clients as a result of co-evolution. Preliminary analysis of RbcX in Chlamydomonas indicated that the protein functions as a Rubisco assembly chaperone in vivo. Therefore, RbcX was silenced using RNAi in Chlamydomonas which resulted in a photosynthetic growth defect in several transformants when grown under light. RbcX mRNA levels were highly decreased in these transformants which resulted in a concomitant decrease of Rubisco large subunit levels. Biochemical and structural analysis from both independent studies in this thesis show that Raf1 and RbcX fulfill similar roles in Rubisco assembly, thus suggesting that functionally redundant factors ensure efficient Rubisco biogenesis.

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Structural und functional characterization of Rubisco assembly chaperones is an episode from Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 06/06 by Ludwig-Maximilians-Universität München.

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This episode was published on Apr 28, 2016.

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